T cell receptor sequences are the dominant factor contributing to the phenotype of CD8+ T cells with specificities against immunogenic viral antigens

SUMMARY Antigen-specific CD8+ T cells mediate pathogen clearance. T cell phenotype is influenced by T cell receptor (TCR) sequences and environmental signals. Quantitative comparisons of these factors in human disease, while challenging to obtain, can provide foundational insights into basic T cell biology. Here, we investigate the phenotype kinetics of 679 CD8+ T cell clonotypes, each with specificity against one of three immunogenic viral antigens. Data were collected from a longitudinal study of 68 COVID-19 patients with antigens from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), cytomegalovirus (CMV), and influenza. Each antigen is associated with a different type of immune activation during COVID-19. We find TCR sequence to be by far the most important factor in shaping T cell phenotype and persistence for populations specific to any of these antigens. Our work demonstrates the important relationship between TCR sequence and T cell phenotype and persistence and helps explain why T cell phenotype often appears to be determined early in an infection.


Figure S1 (
Figure S1 (related to Figure 1): TCR clusters of YLQ-specific CD8 + T cell clonotypes distinguished by unique TCR a and b sequences and GLIPH motifs (A) TCR a motif plots for each cluster.TCR clusters are colored on the TCR-derived UMAP at the center of the plot with arrows connecting each motif plot to their respective location on the UMAP.(B) TCR b motif plots for each cluster.TCR clusters are colored on the TCR-derived UMAP at the center of the plot with arrows connecting each motif plot to their respective location on the UMAP.(C) GLIPH motif plots for each cluster.TCR clusters are colored on the TCR-derived UMAP at the center of the plot with arrows connecting each motif plot to their respective location on the UMAP.

Figure S2 (
Figure S2 (related to Figure 1): Interactions between transcriptomic signatures, TCR clusters, and SARS2-YLQ pMHC-tetramer derived TCRs from literature (A) Heatmap with rows as YLQ-specific TCR-derived clusters and columns as transcriptomic signatures with values scaled per column, legend on bottom.(B) Table of IFNG-producing TCRs and their respective genes and donors are shown on the left.These sequences are mapped to this study's dataset and matched cells are colored on the right on the TCR-derived UMAP.(C) Heatmap with rows as individual genes and columns as TCR-derived clusters with values scaled per row.Left heatmap is for CMV-specific cells and right heatmap is for Flu-specific cells, legend on bottom.

Figure S3 (
Figure S3 (related to Figure 2): Description of RNA UMAP through mRNA, transcriptomic signatures, and comparisons with TCR sequence information (A) Select mRNA transcripts are plotted on the RNA derived UMAP, legend on bottom.(B) Transcriptomic signatures are plotted on the RNA derived UMAP, legend on bottom.(C) Correlation matrix of the positions of TCR clusters in transcriptomic space (RNA UMAP) and in TCR space (TCR UMAPs), legend on right.(D) Comparison of the TCR sequence similarity of the TCR-derived clusters analyzed in depth in Fig. 2 with literature-derived TCR sequences of IFNG-producing YLQspecific CD8 + T cell clonotypes.

Figure
Figure S4 (related to Figures 3 and 4): Phylogenetic tree of the CDR3 regions of contraction correlated TRAVs (A) Dendrogram of a phylogenetic tree computed by MUSCLE describes the relationship between the CDR3 regions of TRAV genes that significantly correlated with the contraction levels of each TCR cluster, legend on bottom.(B) For SARS2-YLQ specific CD8 + T cells after the removal of MAIT cells from the dataset.Bar plot of each -omic on the x-axis and the total number of observed minus expected significant correlations with unbiased PCA dimensions summed for each -omic on the y-axis; negative bars indicate fewer significant correlations with T cell phenotype than statistically expected while positive bars indicate more significant correlations with phenotype than expected.(C) Clustered heatmap of pair-wise TCR distances where lighter colors indicate increased TCR sequence similarity and darker colors indicate pairs with dissimilar TCR sequences.Annotated colors above and to the left of the heatmap indicate functional activity with secretion quantified through ELISA, see upper left legend.Cluster colors indicate the two TCR clusters that emerge from this analysis (cluster A in blue and cluster B in orange).(D) TCR CDR3 a (upper) and b (lower) motifs for TCR cluster A from panel C. (E) Violin plots with the X-axis as the TCR cluster groups from panel C and the Yaxis as normalized functional activity which is secretion measured via ELISA.Each dot is a clonotype, derived by cloning RLIT-specific HLA-A*02:01 restricted TCRs into primary CD8 + T cells from healthy donors.

Figure
Figure S5 (related to Figure 5): TCR sequences separate two distinct bystander activated phenotypes(A) Bar plot with X-axis as a given TCR gene, the gene is labeled on the y-axis, the values for the y-axis is the log2(fold change) in the percentage of the given TCR's prevalence in clonotypes from bystander activated cells with high group 1 versus high group 3 mRNA signatures.(B) TCR a chain sequence motif plots derived from clonotypes from bystander activated cells with high group 1 mRNA signatures on the top and high group 3 mRNA signatures on the bottom.